Sample extraction apparatus with micro elution bed design

ABSTRACT

An apparatus for extracting an analyte from a liquid sample having a container with an entrance, an exit, and a passage therebetween for passage of a liquid sample containing an analyte, the container having a full diameter bed region and a reduced diameter bed region. The container includes a layered construction extending across the passage, having from top to bottom one or more of: (i) an upper flow distributor/support layer, (ii) an upper compression layer, (iii) an extraction layer of microparticulate extraction medium adjacent to the layer (ii), and (iv) a lower compression layer located adjacent to the extraction layer (iii), optionally including one or more air gap layers. At least some of the layers are located in the full diameter bed region, and some of the layers are located in the reduced diameter bed region. The apparatus may have a plurality of containers arranged in an array and/or in series so as to provide multi-stage filtration or extraction.

This is a continuation-in-part application and so claims the benefitpursuant to 35 U.S.C. § 120 of a prior filed and co-pending U.S.Non-Provisional patent application Ser. No. 14/718,065 filed May 20,2015, and entitled “Sample Extraction Apparatus with Micro Elution BedDesign,” which itself claims the benefit of priority pursuant to 35U.S.C. 119(e) to U.S. Provisional Patent Application Ser. No.62/000,759, filed on May 20, 2014, the contents of each of whichapplications are hereby incorporated by reference in their entirety.

The present invention relates to microcolumns for extraction of ananalyte from a liquid sample, and particularly extraction of an analytefrom biological fluids.

Accurate and inexpensive detection of analytes present in liquidsamples, for example in biological fluids, such as blood and urine, isimportant to health care. Tests for analytes in blood and urine areconducted to monitor the health of patients, detect the presence ofdisease conditions, and monitor for the use of illegal or restricteddrugs. For example, doctors, when administering drugs such asanti-arrythymics, asthmatic drugs, insulin, and anticoagulants, checkthe drug content of the blood to regulate the dosages of the patient.Drugs that can be abused, such as heroin, marijuana, cocaine, andcodeine, can be tested to determine abuse of the drug, such as byemployees and by athletes.

A technique used for detection of analytes includes selectivelyextracting the analyte from the biological fluid onto a solid media. Theanalyte is then removed from the solid media by a suitable elutionliquid, and tests are conducted to determine whether the analyte ispresent in the eluent liquid. These tests are conducted using GasChromatography-Mass Spectrometry or Liquid Chromatography-MassSpectrometry.

Extraction columns have been used in the past. For example, particulatesilica has been used as the solid media in a column. In addition, mediahas been sandwiched between frits in a column with a single diametercylindrical shape. Although these prior art devices can be effective, itis desirable to improve on these devices, and their impact in theoverall process and their downstream environmental impact. It isdesirable that the extraction device improve process throughput, removea very high percentage of the analyte from the sample, be transportable,storable without damage, and be inexpensive. Moreover, it is desirablethat any such device be compatible with existing automated equipment,and not leach into the biological or other fluid samples the eluentliquid or any compound that could interfere with the analytical results.Likewise, it is desirable to minimize the media bed volume andassociated dead volumes to lower the volume of the wash eluent liquid.By minimizing the liquid volume, a more concentrated sample is obtainedfor analysis, and the sensitivity of the test is enhanced. High yieldsfrom the sample fluid with minimum elution volumes can be obtained bymaintaining uniform flow through the extraction media, with nochanneling and no dead volume.

SUMMARY

Embodiments of the present invention include an apparatus for extractionof an analyte from a liquid sample. The apparatus includes a containerhaving an entrance, an exit, and a passage therebetween for passage ofthe liquid sample containing an analyte therethrough, said containerhaving a full diameter bed region and a reduced diameter bed region. Theapparatus includes a layered construction having a top and a bottomextending across the passage, comprising from top to bottom: (i) anupper flow distributor/support layer, (ii) an upper compression layer,(iii) an extraction layer of microparticulate extraction medium adjacentto the layer (ii), and (iv) a lower compression layer located adjacentto the extraction layer (iii), where the (i) upper flow distributer and(ii) upper compression layer are located in the full diameter bedregion, the (iii) extraction layer and (iv) lower compression layer arelocated in the reduced diameter bed region. In additional embodiments,the apparatus may include a seven layered construction including (i) anupper flow distributor, (ii) an upper compression layer, (i′) a middleflow distributor, (ii′) a middle compression layer in the reduceddiameter bed region, (iii) an extraction layer of microparticulateextraction medium adjacent to the layer (ii′), (iv) a lower compressionlayer adjacent to layer (iii), and (v) optionally, a lower flowdistributor, wherein layers (i), (ii), (i′), and (ii′) are located inthe full diameter bed region, and layers (iii)-(v) may be located in thereduced diameter bed region.

In one embodiment, the apparatus has one or more air gap layers. In oneembodiment an air gap layer is located in the reduced-diameter bedregion. The air gap layer of a further embodiment has a height thatranges from ½ the diameter of the reduced diameter bed region to 4 timesthe diameter of the reduced diameter bed region. In yet a furtherembodiment, an air gap layer is located between layer (i′) and layer(ii′).

The ratio between the effective area of the full diameter bed region andthe reduced diameter bed region may also vary. The effective area of thefull diameter bed region is A_(F)=π_(F) ², where r_(F) is the radius ofan interior surface of the container in the full diameter bed region,and the effective area of the reduced diameter bed region is A_(r)=π_(r)² where r_(r) is the radius of an interior surface of the container inthe reduced diameter bed region. The ratio between the effective bedarea of the full diameter bed region and the effective area of thereduced diameter bed region ranges from about 10:1 to 1.5:1. In oneembodiment the ratio between the effective area of the extraction mediato the effective area of the upper compression layer is about 1:10. In afurther embodiment the ratio between the effective area of theextraction media to the effective area of the upper compression layer isabout 1:4. In another embodiment, the ratio between the effective areaof the full diameter bed region and the effective area of the reduceddiameter bed region is about 4:1.

The extraction media may also be tailored to work with a particularanalyte. In one embodiment the extraction media has a number averageparticle size of about less than 20 μm. In a further embodiment, theextraction media has a number average particle size of about less than10 μm.

The present apparatus may have a plurality of containers arranged in anarray, optionally having a collection plate with a corresponding arrayof wells.

In a further embodiment, the apparatus has dual barrels or is configuredin two stages, again whether with a single container or multiplecontainers per stage. The top barrel or first stage is equipped with afiltration system to remove unwanted impurities as the analyticalsamples are passed through the top barrel. Upon passing through the topbarrel, the analytes of interest may be caught by the bottom barrel orsecond stage column filled with desired amount of sorbent, enablingrelatively cleaner extracts with relatively smaller elution volumes.

Further embodiments of the invention are directed to methods of usingthe present apparatus, and kits including the present apparatus.

The present invention and its multiple embodiments are described furtherbelow. Of course, variations on these described embodiments will becomeapparent to those of ordinary skill in the art upon reading the presentdescription, and thus, one of skill in the art would recognize suitablecombinations and variations.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIG. 1A is a schematic perspective view of the exterior of the presentapparatus. FIG. 1B is an enlarged schematic cross-sectional side view ofone embodiment of the layers used in the present apparatus.

FIG. 2A is a cross-sectional side view of a conventional microcolumn M.FIG. 2B is a cross-sectional side view of an alternative embodiment ofthe present apparatus. FIG. 2C is a cross-sectional side view of afurther alternative embodiment of the present apparatus with one or moreribs 50 on the exterior. FIG. 2D is a partial perspective view of thelower narrow diameter portion of the present apparatus, showing the ribsused in a luer tip system. FIG. 2E is an exterior side view of thepresent apparatus including the luer tip at the exit end thereof.

FIG. 3 is a schematic cross-sectional side view of a further alternativeembodiment of the layers used in the present apparatus which include anair gap.

FIG. 4A is exemplary data obtained with the present apparatus using 10pg/ml of dopamine. FIG. 4B is exemplary data obtained with the presentapparatus using 10 pg/ml of dopamine. FIG. 4C is exemplary data obtainedwith the present apparatus using 10 pg/ml of norepinephrine. FIG. 4D isexemplary data obtained with the present apparatus using 10 pg/ml ofnorepinephrine. FIG. 4E is exemplary data obtained with the presentapparatus using 10 pg/ml of epinephrine. FIG. 4F is exemplary dataobtained with the present apparatus using 10 pg/ml of epinephrine.

FIG. 5A is exemplary data obtained with the present apparatus using 100pg/ml dopamine. FIG. 5B is exemplary data obtained with the presentapparatus using 100 pg/ml of dopamine. FIG. 5C is exemplary dataobtained with the present apparatus using 100 pg/ml of norepinephrine.FIG. 5D is exemplary data obtained with the present apparatus using 100pg/ml of norepinephrine. FIG. 5E is exemplary data obtained with thepresent apparatus using 100 pg/ml of epinephrine. FIG. 5F is exemplarydata obtained with the present apparatus using 100 pg/ml of epinephrine.

FIG. 6A is calibration curve of buprenorphine extracted from urine withthe present apparatus. FIG. 6B is calibration curve of norbuprenorphineextracted from urine with the present apparatus.

FIG. 7A is a schematic cross-sectional side view of a furtheralternative embodiment of the layers used in the present apparatus whichinclude an air gap. FIG. 7B is a schematic cross-sectional side view ofa further alternative embodiment of the layers used in the presentapparatus which include multiple air gaps.

FIG. 8A is a schematic cross-sectional side view of a furtheralternative embodiment of the layers used in the present apparatus hereincluded only in the upper full diameter bed region. FIG. 8B is aschematic cross-sectional side view of a further alternative embodimentof the layers used in the present apparatus again included only in theupper full diameter bed region, with an optional frit layer.

FIG. 9A is a schematic cross-sectional side view of a furtheralternative embodiment of the layers used in the present apparatus hereincluded only in the intermediate reduced diameter bed region. FIG. 9Bis a schematic cross-sectional side view of a further alternativeembodiment of the layers used in the present apparatus again includedonly in the intermediate reduced diameter bed region, with an optionalair gap.

FIG. 10A is a perspective view of an exemplary embodiment of the presentapparatus having dual barrels or configured in two stages. FIG. 10B isan enlarged partial cross-sectional side view of the upper barrel orfirst stage thereof. FIG. 10C is an enlarged partial cross-sectionalside view of the lower barrel or second stage thereof.

FIG. 11A is a perspective view of a further exemplary embodiment of thepresent apparatus having dual barrels or configured in two stages. FIG.11B is an enlarged partial cross-sectional side view of the upper barrelor first stage thereof. FIG. 11C is an enlarged partial cross-sectionalside view of the lower barrel or second stage thereof.

DETAILED DESCRIPTION

The present invention is directed to an extraction apparatus that meetsthe needs of: improving downstream environmental impact and processthroughput; removing a very high percentage of the analyte from thesample; transportability; storage without damage; price point;compatibility with existing automated equipment; leachingcharacteristics; minimization of the media bed volume and associateddead volumes; and enhancing sensitivity and uniformity of flow throughthe extraction media.

The present apparatus is useful for extracting an analyte from a liquidsample and comprises a container, typically a microcolumn, having anentrance, an opposed exit, and a passage therebetween for passage of aliquid sample containing an analyte therethrough.

Referring to FIGS. 1A and 1B, the microcolumn 12 of the presentapparatus 10 has at least two regions in the passage 23 of the analyte:an area having a full diameter bed (aka the “full diameter bed region”,shown in FIGS. 1A and 1B as 30) and an area having a reduced diameterbed (aka the “reduced diameter bed region”, shown in FIGS. 1A and 1B as31). As used herein, “diameter bed area” is measured by the surface areaof a horizontal cross-section of the interior cavity of the container.Therefore, for a cylindrical container the diameter bed area is the areaof the circle (πr²) whose diameter (=2 r) extends from one side of theinterior cavity of the container to the other side of the interiorcavity. The diameter bed area may also be referred to as the “effectivearea” of a particular layer.

Within the passage 23 in the reduced diameter bed region 31 is a layerof a microparticulate extraction media. Extraction media 14 may includeany known sorbent and a variety of particles. The sorbent particlesemployed in the apparatus include any particulate matter that is capableof having at least one substance, either target or interfering, adheredthereto. Illustrative examples of sorbent particles that may be employedin the present invention include, but are not limited to: ion exchangesorbents; reversed phase sorbents; and normal phase sorbents. Moreparticularly, the sorbent particles may be an inorganic material such asSiO₂ or an organic polymeric material such as poly(divinylbenzene). Insome embodiments of the present invention, the sorbent particles may betreated with an organic functional group such as a C₂-C₂₂, preferablyC₈-C₁₈ functional group. In one embodiment, the sorbent of theextraction media includes silica based particles (such as silica basedcarboxylic acids), diatomaceous earth particles, polymeric basedparticles, mono-dispersed silica and polymeric particles, and/or carbongraphite particles. The media is selected for extracting the analytefrom the liquid sample. A suitable silica extraction media is describedin U.S. Pat. No. 4,650,714, which is incorporated herein by reference. Apreferred microparticulate silica extraction media is available fromAvantor Performance Materials (previously known as J.T. Baker ChemicalCompany) of Phillipsburg, N.J., and is sold under their catalog number7049-01.

The extraction media 14 has a small particle size having a numberaverage particle size of less than about 40 microns, less than about 30microns, less than about 25 microns, less than about 20 microns, lessthan about 15 microns, less than about 10 microns, or less than about 5microns. In one embodiment, the extraction media has a number averageparticle size of less than about 20 microns, or more preferably lessthan about 10 microns. In addition, the extraction media does not haveto be homogenous, but rather a different extraction media can be used ina single bed, or the apparatus can include multiple beds of extractionmedia for extracting different analytes from samples.

The extraction media 14 is sandwiched between at least two compressionlayers. In one embodiment, the diameter bed area of the extraction mediais lower than the diameter bed area of the upper compression layer, suchthat the ratio of the effective area of the upper compression layer tothe effective area of the extraction media layer is about 10 to 1, about9 to 1, about 8 to 1 about 7 to 1 about 6 to 1, about 5 to 1, about 4 to1, about 3 to 1, about 2 to 1, or about 1.5 to 1. In one embodiment, theratio of the effective area of the upper compression layer to theeffective area of the extraction media layer is about 4 to1.

The extraction media may be loosely packed (where the sorbent is looselysandwiched between the compression layers and free to move) or acompacted extraction media bed (where the extraction media is compactedbetween the two layers, or has relatively little additional possiblespace between particles).

The extraction media is sandwiched between either the upper compressionlayer 18 a and lower compression layer 18 b or the middle compressionlayer 18 c and lower compression layer 18 b, and compress the extractionmedia there between. Upper compression layer 18 a is located in fulldiameter bed region 30, middle compression layer 18 c may be located ineither the full diameter bed region 30 or reduced diameter bed region31, and lower compression layer 18 b is located in reduced diameter bedregion 31. In one embodiment only two compression layers are used. Inone embodiment, at least three compression layers are used.

The compression layers are sufficiently porous that the liquid samplecan flow therethrough, and are formed from a flexible material. One ormore of the compression layers may be flat, spherical, or optimallyshaped to suit the fluid flow through the apparatus (such as a truncatedcone, prismatic, truncated pyramid, etc.). FIG. 3 shows an embodimentwhere the middle and lower compression layers (18 c and 18 b,respectively) are spherical. A mixture of shapes of compression layersmay be used in a single microcolumn.

The narrow bore sandwich design pairs with the low dead volume columndesign for function. The chief purpose of compression layers 18 is tohold the extraction media 14 in place and compressed as a thinextraction layer. In one embodiment, one or more of the compressionlayer(s) has a pore size less than the particle size of the extractionmedia and functions as a flow rate limiter. They are sufficiently porousthat the liquid sample can flow therethrough, and are composed of aflexible, hydrophilic material. The compression layer is preferablyformed of a spongy, glass fiber, having no binder.

A suitable compression layer comprises a glass microfiber media made ofanalytically clean material. Suitable materials, which are availablefrom Whatman Specialty Products, Inc. of Fairfield, N.J., includeborosilicate glass fibers that are analytically clean and include nobinder. This material, when purchased, has a smooth side and a roughside, where the smooth side is of lower porosity than the rough side.Preferably, it is the smooth side that is placed in contact with themicroparticles of extraction layer 14. Other suitable materials includefrits and filters, such as polymer (e.g., polypropylene or polyethylene)frit materials. Said frits or filters may be cylindrical, die cut, orspherical, having a diameter to fit the interior cavity of the column;for instance, in the full diameter portion or in the reduced diameterportion.

Preferably compression layers 18 are resilient or “spongy” to hold themicroparticles in place. In one aspect, the pore size for thecompression layers is less than 10 microns, less than 5 microns, or lessthan 3 microns. Compression layers 18 generally are of the samethickness, having a thickness typically of from about 0.1 mm to about3.25 mm, about 0.25 mm to about 3.25 mm, about 0.5 mm to about 3.0 mm,about 0.75 mm to about 3 mm, about 0.25 mm to about 2.5 mm, about 0.25mm to about 2 mm, about 0.25 mm to about 1.5 mm, about 0.25 mm to about1.25 mm, about 0.25 mm to about 1.0 mm, about 0.1 mm to about 0.75 mm,or about 0.1 mm to about 0.5 mm. In one aspect the compression layer(s)have a thickness of about 0.5 mm.

This is in contrast to the prior art microelution columns, which aremade with cylindrical syringe barrel tubes, or funnel shaped samplereservoirs (see e.g., US 2006/0163163). Although some of thesemicroelution columns have reduced dead volume after the bed, thesedesigns do not address the ratio between the effective area of the uppercompression layer compared to the effective area of the extraction medialayer.

Reducing the effective area (πr²) of the extraction media bed about 4-1in relation to the effective area of the upper compression layer leadsto a corresponding reduced volume of sorbent material and reduced deadvolumes for the reagent.

Flow distributors 16, which are formed of a flexible mesh material, helpprovide uniform flow of the sample through the column, and physicallyretain the compression layers and microparticulate material in place inthe column. Preferably, the mesh is 200 mesh or smaller, (i.e., has amesh number of 200 or higher). In aspects of this embodiment, the meshnumber is 150 or higher, 170 or higher, 200 or higher, 250 or higher,270 or higher, 325 or higher, or 400 or higher. The mesh may be made ofany flexible bio-inert material. It one embodiment it is made ofPolyphenelyne Sulfide (PPS), Polytetrafluoroethylene (PTFE), Polyetherether Ketone (PEEK), Polyoxymethylene (POM), Ethylene Propylene Diene(EPDM), a Fluorinated elastomer (FKM), a Perfluoro elastomer (FFKM),polysulfone (PSU), Ethylene Tetrafluoroethylene (ETFE), Polypropylene(PP), (Poly)Chlorotrifluoroethylene (PCTFE/CTFE), polystyrene, highdensity polyethylene, polycarbonate, nylon, polyethylene terephthalate(PET), silicon, rubber, or polyester. In aspects of this embodiment itis made of polypropylene, or alternatively, polytetrafluoroethylene. Asuitable material is available from Tetko, Inc. of Briarcliff Manor,N.Y., under catalog number 5-420134.

In one embodiment the microcolumn 12 also includes an upper mesh flowdistributor 16 a above the upper compression layer 18 a for support,and, optionally, a middle flow distributor 16 b and/or a lower flowdistributor 16 c. In one embodiment, the flow distributors may belayered or molded in the housing above and below the compressionlayer(s), sandwiching the compression layers 18 and the layer ofextraction media 14 therebetween. The flow distributors 16 hold theextraction media 14 and the compression layers 18 in the reduceddiameter bed region 31 of the microcolumn 12 and help distribute flow ofthe liquid sample to avoid channeling. As shown in the embodiment ofFIG. 1B, the upper flow distributor 16 a is located in the full diameterbed region 30, the middle flow distributor 16 b is also located in thefull-diameter bed region 30 and the lower flow distributor 16 c, ifused, seats in the lower portion of the column in the reduced diameterbed region 31. In one embodiment, the upper flow distributor 16 a issized so that it is held in the bore of microcolumn 12 by a compressionfit. Similarly, the other flow distributor layer(s) may also be sizedfor a compression fit.

Due to the combination of the narrow bore extraction media and thecompression layer sandwich, rapid extraction of an analyte from a fluidcan be obtained, with the apparatus being configured for very smallvolumes of elution liquid on the order of about 0.025 mL to about 0.25mL, about 0.025 mL to about 0.2 mL, about 0.025 mL to about 0.15 mL,about 0.025 mL to about 0.100 mL, down from 0.5 mL to 1.5 mL. Thissmaller elution volume fits directly into autosampler trays forautomation. Smaller volumes eliminate concentration steps and vialtransfers and associated cross contamination fails. In addition, theextraction device of the present invention is inexpensive to use andmanufacture, is stable during storage and transportation, and iscompatible with existing automated equipment. The experimental databelow displays the effective improvement in performance of a clinicalassay done using regular Cerex columns versus the Narrow Bore version(the presently disclosed columns).

An apparatus 10 for extracting an analyte from a liquid sample is againshown in FIG. 1A. As shown in FIG. 1A, the apparatus 10 comprises themicrocolumn 12, which serves as a container for an extraction sandwichsystem. In one embodiment, microcolumn 12 has generally a tubularconfiguration, and has entrance 20, opposed exit 22, and passage 23therebetween. Passage 23, which is also referred to as a central bore,contains the extraction system. Passage 23 has two regions, upper fulldiameter bed region 30 and lower reduced diameter bed region 31. Theexit 22 may optionally be located in a separate region 33, with a “tip”configuration.

The various layers may be located adjacent to each other, and may or maynot be in direct contact with the surrounding layers. That is, withreference to FIG. 3, there may be one or more Air Gaps 301 between twolayers which are deliberately located to prevent dripping or capillaryflow and allow for the transfer of the column to an appropriatereceiving vessel or plate. For instance, in one embodiment, there is anAir Gap layer 301 between the middle flow distributor 16 b and themiddle compression layer 18 c. That is, in one embodiment, there may bean air gap between the layers that straddle the transition from the fulldiameter bed region 30 to the reduced diameter bed region 31. In anotherembodiment there may be a strategic Air Gap 301 located after the lowercompression layer 18 b but before the optional lower distributor layer16 c.

In one embodiment, the Air Gap is positioned at or below the top edge ofthe reduced diameter bed region, and may have a height ranging from ½ ofthe diameter of the reduced diameter bed region to 4 times the diameterof the reduced diameter bed region. In aspects of this embodiment, theheight of the air gap may be from, e.g., about ½ to about 1 times, about½ to about 1.5 times, about ½ to about 2 times, about ½ to about 2.5times, about ½ to about 3 times, about % to about 3.5 times, about % toabout 4 times, about 1 to about 1.5 times, about 1 to about 2 times,about 1 to about 2.5 times, about 1 to about 3 times, about 1 to about3.5 times, about 1 to about 4 times, about 1.5 to about 2 times, about1.5 to about 2.5 times, about 1.5 to about 3 times, about 1.5 to about3.5 times, about 1.5 to about 4 times, about 2 to about 2.5 times, about2 to about 3 times, about 2 to about 3.5 times, about 2 to about 4times, about 2.5 to about 3 times, about 2.5 to about 3.5 times, about2.5 to about 4 times, about 3 to about 3.5 times, about 3 to about 4times, or about 3.5 to about 4 times, the diameter of the reduceddiameter bed region.

When placed strategically at the inlet to the reduced diameter bedregion, the air gap prevents the unassisted capillary flow of certainclasses of solvents or samples from bridging the gap for capillarytransfer down to the next layer and then through the media bed. The airgap is particularly useful in areas with smaller diameters because thestrength of the air gap is based on the surface tension between theliquid flowing through the apparatus and the gas located in the air gap.

The gap height may vary by the intended analytes to be tested, and mightnot be there at all. The Air Gap is particularly useful in a methodwhere the compression layers are wet or preconditioned before the testsample is added to the microcolumn.

The structure of tip region 33 is not particularly limited and in oneembodiment may be cylindrical or conical. Tip region 33 may have thesame diameter as the reduced diameter bed region 31 or a smallerdiameter than the reduced diameter bed region 31. In one embodiment, thetip region 33 has the same footprint as the reduced diameter bed region31; in another embodiment, the footprint of the tip region 33 has adifferently shaped footprint than the reduced diameter bed region 31. Inone embodiment, tip region 33 is optionally in the form of a luer tip orsimilar, which allows apparatus 10 to be used with conventionalautomated extraction apparatuses, which are designed to receive anextraction column having a luer tip.

In another embodiment, FIG. 2C provides a vertical cross-section of thepresent apparatus 10 with one or more ribs 50 on the exterior, thoughwith all interior layers or features removed for simplicity. FIG. 2D isa partial perspective view of the lower narrow diameter portion of thepresent apparatus 10, showing the ribs 50 used in a luer tip system.FIG. 2E is a side view of the present apparatus 10 including the luertip system at the exit end of the present apparatus.

A liquid sample flows in the direction of arrow 26 shown in FIG. 1Bthrough the passage 23.

The portion of microcolumn 12 above the extraction sandwich systemserves as a reservoir for the liquid sample, from which an analyte is tobe extracted, and also a reservoir for a washing liquid, and an eluentliquid.

In one embodiment, the extraction system includes a four-layer sandwichconstruction with: (i) an upper flow distributor/support, (ii) an uppercompression layer, (iii) an extraction layer, and (iv) a lowercompression layer, where the upper flow distributer is located in thefull diameter bed region and the upper compression layer, the extractionlayer and the lower compression layer are located in the reduceddiameter bed region.

In one embodiment, the extraction system is comprised of a five-layersandwich construction, including (i) an upper flow distributor, (ii) acylindrical or fabric compression layer, (iii) a spherical orcylindrical frit as a compression layer, (iv) the microparticulateextraction medium, and (v) a spherical or cylindrical frit as a lowercompression layer. Layers (i)-(ii) would reside in the full diameter bedregion, where layers (iii)-(v) would reside in the reduced diameter bedregion.

In one embodiment, the extraction system is comprised of a six-layersandwich construction, including (i) an upper flow distributor, (ii) acylindrical or fabric compression layer, (iii) a lower flow distributor,(iv) a spherical or cylindrical frit as a compression layer, (v) themicroparticulate extraction medium, and (vi) a spherical or cylindricalfrit as a lower compression layer. Layers (i)-(iii) would reside in thefull diameter bed region, where layers (iv)-(vi) would reside in thereduced diameter bed region. In one embodiment as shown in FIG. 1B, theextraction system is comprised of a seven-layer sandwich construction,that includes (i) upper flow distributor 16 a, (ii) upper compressionlayer 18 a, (iii) middle flow distributor 16 b, (iv) middle compressionlayer 18 c in the reduced diameter bed region 31, (v) extraction layer14 of microparticulate extraction medium, (vi) lower compression layer18 b, and optionally (vii) lower flow distributor/support 16 c which maybe molded as part of the container. In this embodiment, layers (i)-(iii)may be located in the full diameter bed region, and layers (iv)-(vii)may be located in the reduced diameter bed region. Alternatively, thedivision between full diameter and reduced diameter may occur betweenlayers (ii) and (iii). Additional layers may be added.

In a further embodiment as shown in FIG. 3, the extraction system iscomprised of an eight-layer sandwich construction, that includes: (i)upper flow distributor 16 a, (ii) upper compression layer 18 a, (iii)middle flow distributor 16 b, (iv) air gap layer 301, (v) middlecompression layer 18 c in the reduced diameter region 31, (vi)extraction layer 14 of microparticulate extraction medium, (vii) lowercompression layer 18 b, and optionally (viii) lower flowdistributor/support 16 c which may be molded as part of the container.In this embodiment, layers (i)-(iii) may be located in the full diameterbed region, and layers (iv)-(viii) may be located in the reduceddiameter bed region. Alternatively, the division between full diameterand reduced diameter may occur between layers (ii) and (iii). Additionallayers may be added.

In a still further embodiment as shown in FIG. 7A, the extraction systemis comprised of a seven-layer sandwich construction, that includes: (i)upper flow distributor 16 a, (ii) upper compression layer 18 a, (iii)middle flow distributor 16 b, (iv) middle compression layer 18 c in thereduced diameter region 31, (v) air gap layer 314 in the place ofextraction layer 14 (FIGS. 1B and 3), (vi) lower compression layer 18 b,and optionally (vii) lower flow distributor/support 16 c which may bemolded as part of the container. In this embodiment, layers (i)-(iii)may be located in the full diameter bed region, and layers (iv)-(vii)may be located in the reduced diameter bed region. Alternatively, thedivision between full diameter and reduced diameter may occur betweenlayers (ii) and (iii). Additional layers may be added.

In a still further embodiment as shown in FIG. 7B, the extraction systemis comprised of an eight-layer sandwich construction, that includes: (i)upper flow distributor 16 a, (ii) upper compression layer 18 a, (iii)middle flow distributor 16 b, (iv) air gap layer 301, (v) middlecompression layer 18 c in the reduced diameter region 31, (vi) air gaplayer 314 in the place of extraction layer 14 (FIGS. 1B and 3), (vii)lower compression layer 18 b, and optionally (viii) lower flowdistributor/support 16 c which may be molded as part of the container.In this embodiment, layers (i)-(iii) may be located in the full diameterbed region, and layers (iv)-(viii) may be located in the reduceddiameter bed region. Alternatively, the division between full diameterand reduced diameter may occur between layers (ii) and (iii). Additionallayers may be added.

In connection with the alternative embodiments of FIGS. 7A and 7B, theremay again be one or more Air Gaps 301, 314 between two layers which aredeliberately located to prevent dripping or capillary flow and allow forthe transfer of the column to an appropriate receiving vessel or plate.For instance, in one such embodiment, there is an Air Gap layer 314between the middle compression layer 18 c and the lower compressionlayer 18 b in the place of the extraction layer 14 (FIGS. 1B and 3),though it could be in addition to such extraction layer 14. Or there isan Air Gap layer 301 between the middle flow distributor 16 b and themiddle compression layer 18 c as well, so as to have effectively two airgaps separated by at least one material layer, further contributing tothe prevention of dripping or capillary flow. That is, in oneembodiment, there may be an air gap between the layers that straddle thetransition from the full diameter bed region 30 to the reduced diameterbed region 31 and/or between the layers of the reduced diameter bedregion 31. In any such embodiments the height of thickness of anyparticular air gap, just as the material layers, can vary depending onthe context, such that all drawings are to be understood as illustrativeand not to scale and thus non-limiting.

In a still further embodiment as shown in FIG. 8A, the extraction systemis comprised of a three-layer sandwich construction, that includes: (i)upper flow distributor 16 a, (ii) upper compression layer 18 a, and(iii) middle flow distributor 16 b. In this embodiment, layers (i)-(iii)may be located in the full diameter bed region, with no layers locatedin the reduced diameter bed region. Alternatively, the division betweenfull diameter and reduced diameter may occur between layers (ii) and(iii). Additional layers may be added.

In a still further embodiment as shown in FIG. 8B, the extraction systemis comprised of a four-layer sandwich construction, that includes: (i)upper flow distributor 16 a, (ii) upper compression layer 18 a, (iii)optional frit 17, and (iv) middle flow distributor 16 b. In thisembodiment, layers (i)-(iv) may be located in the full diameter bedregion, with no layers located in the reduced diameter bed region.Alternatively, the division between full diameter and reduced diametermay occur between layers (iii) and (iv). Additional layers may be added.

According to the alternative exemplary embodiments of FIGS. 8A and 8B,it will be appreciated that microcolumns 12 are shown having a first orupper extraction system or layered construction located in the upperfull diameter bed region but no extraction system or layers in the lowerreduced diameter bed region in which solid phase extraction or the likewould have been conducted as in other embodiments herein. Such a reducedextraction system would have other clinical uses in single or dual stageapplications, such as described below in connection with FIG. 10.

In a still further embodiment as shown in FIG. 9A, the extraction systemis comprised of a four-layer sandwich construction, that includes: (i)middle compression layer 18 c in the reduced diameter region 31, (ii)extraction layer 14 of microparticulate extraction medium, (iii) lowercompression layer 18 b, and optionally (iv) lower flowdistributor/support 16 c which may be molded as part of the container.In this embodiment, layers (i)-(iv) may be located in the reduceddiameter bed region, with no layers located in the full diameter bedregion. Additional layers may be added.

And in a still further embodiment as shown in FIG. 9B, the extractionsystem is comprised of a four-layer sandwich construction, thatincludes: (i) middle compression layer 18 c in the reduced diameterregion 31, (ii) air gap layer 314 in the place of extraction layer 14(FIG. 9A), (iii) lower compression layer 18 b, and optionally (iv) lowerflow distributor/support 16 c which may be molded as part of thecontainer. In this embodiment, layers (i)-(iv) may again be located inthe reduced diameter bed region, with no layers located in the fulldiameter bed region. Additional layers may be added.

According to the alternative exemplary embodiments of FIGS. 9A and 9B,it will be appreciated that microcolumns 12 are shown having a second orlower extraction system or layered construction located in the lowerreduced diameter bed region but no extraction system or layers in theupper full diameter bed region in which the upper or middle flowdistributor layers 16 a, 16 b or the like would have been positioned asin other embodiments herein. Such a reduced extraction system would haveother clinical uses in single or dual stage applications, such asdescribed below in connection with FIGS. 10 and 11.

All of the components of apparatus 10 are made of materials that aresubstantially inert to biological fluids so that when a biological fluidsuch as blood or urine is passed through apparatus 10, substantiallynothing passes from apparatus 10 into the blood or urine. In oneembodiment, microcolumn 12 is made of a biologically inert material. Inone aspect, the biologically inert material is a plastic. In aspects ofthis embodiment, the biologically inert material is a fluorinatedpolymer or polypropylene. In other aspects the material is PolyphenelyneSulfide (PPS), Polytetrafluoroethylene (PTFE), Polyether ether Ketone(PEEK), Polyoxymethylene (POM), Ethylene Propylene Diene (EPDM), aFluorinated elastomer (FKM), a Perfluoro elastomer (FFKM), polysulfone(PSU), Ethylene Tetrafluoroethylene (ETFE), Polypropylene (PP),(Poly)Chlorotrifluoroethylene (PCTFE/CTFE), polystyrene, high densitypolyethylene, polycarbonate, nylon, or polyethylene terephthalate (PET),silicon, rubber, polyester, or ceramics.

A typical microcolumn according to the present invention has an internaldiameter of about 0.01 inch to about 2 inches, about 0.025 inches toabout 1.75 inches, about 0.05 inches to about 1.5 inches, of about 0.075inches to about 1.25 inches, of about 0.1 inches to about 1 inch. Inother aspects of this embodiment, the internal diameter is at least 0.01inch, at least 0.025 inches, at least 0.05 inches, 0.075 inches, atleast 0.1 inch, at least 0.25 inches, at least 0.5 inches, at least 0.75inches, at least 1 inch, at least 1.25 inches, at least 1.5 inches, atleast 1.75 inches, or at least 2 inches. In still other aspects of thisembodiment, the internal diameter is at most 0.01 inch, at most 0.025inches, at most 0.05 inches, 0.075 inches, at most 0.1 inch, at most0.25 inches, at most 0.5 inches, at most 0.75 inches, at most 1 inch, atmost 1.25 inches, at most 1.5 inches, at most 1.75 inches, or at most 2inches. In a preferred embodiment the microcolumn has an internaldiameter of about 0.1 inch to 1.0 inch.

A typical microcolumn according to the present invention has a length,excluding the tip if present, of about 0.25 inches to about 5 inches,0.5 inches to about 4.5 inches, 0.5 inches to about 4 inches, 0.5 inchesto about 3.5 inches, about 0.5 inches to about 3 inches, about 0.5inches to about 2.5 inches, about 0.5 inches to about 2 inches, about0.5 inches to about 1.5 inches, about 0.5 inches to about 1 inch, about1.75 inches to about 3 inches, about 2 inches to about 3 inches, about2.5 inches to about 3 inches. In other aspects of the invention, themicrocolumn has a length of at least 0.25 inches, at least 0.5 inches,at least 0.75 inches, at least 1 inch, at least 1.25 inches, at least1.5 inches, at least 2 inches, at least 2.25 inches, at least 2.5inches, at least 2.75 inches, at least 3 inches, at least 3.25 inches,at least 3.5 inches, at least 3.75 inches, at least 4 inches, at least4.25 inches, at least 4.5 inches, at least 4.75 inches, or at least 5inches. In still further aspects, the microcolumn has a length,excluding the tip if present of at most 0.25 inches, at most 0.5 inches,at most 0.75 inches, at most 1 inch, at most 1.25 inches, at most 1.5inches, at most 2 inches, at most 2.25 inches, at most 2.5 inches, atmost 2.75 inches, at most 3 inches, at most 3.25 inches, at most 3.5inches, at most 3.75 inches, at most 4 inches, at most 4.25 inches, atmost 4.5 inches, at most 4.75 inches, or at most 5 inches. In apreferred embodiment, the microcolumn has a length, excluding the tip ofabout 0.5 inches to about 3 inches. The length of the tip (if present)is not particularly limited but may range from about 0.1 inch to 1 inch.

The upper full diameter bed region generally includes at least about40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 85% of the totalmicrocolumn length. In one embodiment, the upper full diameter bedregion includes at least about 75% of the total microcolumn length.

The microcolumn of the apparatus need not have the shape shown in thefigures. For example, it need not be cylindrical, and may instead have asquare footprint, a polygonal footprint (e.g., hexagonal, octagonal,etc.), tubular, or include combinations of multiple shapes. As usedherein “footprint” describes the shape of a horizontal cross-section ofthe interior cavity (i.e., passage 23) of the microcolumn. In oneembodiment, the full diameter bed region has a first footprint shape,and the reduced diameter bed region has a second footprint shape, andthe tip (if present) may have a third footprint shape (or may have thesame footprint shape as either the full diameter bed region or thereduced diameter bed region).

In addition, in one embodiment of the invention, entrance 20 can bedesigned or configured to receive a connective fitting to connect themicrocolumn to a fluid input device. Such connective fittings includeluer tips, luer-lock extensions or tapers of various types, male andfemale-type connections of various types, threaded connections, or barbconnections.

As used herein, a “fluid input device” is any device which is in contactwith the entrance and directly transfers the sample or reagent toentrance 20 in a connected manner. Fluid input devices may includeautomated fluid dispensing systems, automated liquid handling platforms,syringes, and microdispensers. The fluid input device may dispense thesample or reagent to the microcolumn automatically, semi-automatically,or manually. For instance a fluid input device may include a reservoircontaining a liquid sample, which once connected to the entrance, canautomatically dispense sample to the microcolumn.

The narrow bore sandwich layer in the reduced diameter region can beassembled as a stand-alone column barrel design or patterned in aone-piece block format for automated processing.

The present apparatus may be in a single column format, which isconvenient and cost effective for preparing a small number of samples,or a multi-column array or format (aka, an “extraction plate”), which issuited for preparing large numbers of samples in parallel.

One embodiment of the invention is an extraction plate configured for orcontaining the narrow bore columns discussed above. This extractionplate may be a molded plate containing a plurality of columns. Thecolumns may be arrayed to align or intercalate with the wells ofconventional “multi-well” formats, such that each column would eluteinto a well in a standard (or custom) multi-well plate. Multi-wellformats are commonly used with robotic fluid dispensing systems, such asautosamplers. Typical multi-well formats are not limited, but include48-, 96-, and 384- and 1,584-well standard plate formats.

Fluids are usually forced through the present apparatus and into thecollection containers (or the wells of a “collection tray” or “plate”),either by drawing a vacuum across the device with a specially designedvacuum manifold, or by using centrifugal or gravitational force. Inspecially designed vacuum manifold systems, both the column and thereceiving means (such as a well or collection tube) may be integratedinto the vacuum system to optimize extraction. Centrifugal force isgenerated by placing the apparatus, together with a suitable collectiontube or tray, into a centrifuge specifically designed for the intendedpurpose. However, in one embodiment gravity may be sufficient to forcethe fluid through the present apparatus as well.

The present apparatus may use conventional collection containers orcollection plates, such as glass tubes, centrifuge tubes, Eppendorftubes, or standard multi-well plates or trays. The present apparatus mayalternately use collection containers specifically designed to becompatible with the present extraction plate.

Methods of using the present invention include extraction of an analytefrom a test sample. A test sample refers to any sample that may containan analyte of interest. A test sample may be a biological sample, thatis, a sample obtained from any biological source, such as an animal, aplant, a fungus, a microorganism, a cell culture, an organ culture, etc.In aspects of this embodiment, a biological sample includes a bloodsample including a whole blood sample, a plasma sample, or a serumsample, a saliva sample, a urine sample, cerebrospinal fluid sample, abile sample, a tissue sample, or any other sample that can be obtained,extracted or isolated from a biological source. Such biological samplesmay be obtained, for example, from a patient; that is, a living person,male or female, presenting oneself in a clinical setting for diagnosis,prognosis, or treatment of a disease or condition. In one embodiment,the sample is obtained from a patient, for example, a plasma specimen.The plasma specimen may be taken with or without the use ofanticoagulants.

A test sample may be an environmental sample. Environmental samples aresamples taken from dirt, plant matter, or fluid sources (such as groundwater, oceans, or rivers etc.). Dirt (aka “soil samples”) may be takenfrom agricultural sites or sites of environmental interest and may havethe analyte extracted, including the removal of particulate matter.

The methods of using the present apparatuses include contacting theapparatus with the sample in a liquid buffer, and eluting the samplefrom the apparatus with an elution buffer. Further steps might includeconditioning the apparatus and washing steps (before, during, or aftercontacting the apparatus with a sample).

The present apparatus may also be included in a kit specificallydesigned to capture and separate a particular analyte of interest. Themicroparticulate extraction media may be specifically adapted toseparate specific analytes of interest. The kit may include suitablereagents (labels, washes, etc.), buffers or elution buffers, inconcentrated form or in a form suitable for direct use. The kit may alsoinclude an extraction plate or array of columns as described above, andaccompanying couplings to connect the plate to fluid dispensing devicesand/or plates or vials for receiving the eluted solution containing thesample.

Turning next to FIGS. 10A-10C there is shown a further exemplaryembodiment of an apparatus 110 for extracting an analyte from a liquidsample, here the apparatus 110 configured as a multi-column array orformat (aka, an “extraction plate”) of microcolumns 112, 212 in seriesas by stacking or nesting one array of microcolumns on or over another,which arrangement is suited for preparing large numbers of samples inparallel and further for multi-stage extraction or elution as describedfurther below. Here, there are thus formed an upper first array 170 offirst microcolumns 112 and a lower second array 270 of secondmicrocolumns 212. In the illustrated embodiment, each array 170, 270comprises a plurality of microcolumns 112, 212 maintained in asubstantially parallel and offset pattern or arrangement as by arespective top plate 160, 260 and base 162, 262—as illustrated, thereare ninety-six (96) such microcolumns 112, 212 per array 170, 270,though it will be appreciated that such is merely exemplary. The nestingof respective first and second microcolumns 112, 212 may be achievedemploying any appropriate technique now known or later developed.However, while a tight or interference fit or substantially sealedarrangement as typically configured may be employed in nesting the firstand second microcolumns 112, 212 in series where the sample is drawnthrough both microcolumns 112, 212 simultaneously as by applyingpressure or a vacuum or by centrifugal or gravitational force, uniquelyaccording to aspects of the present invention a loose or unsealednesting of the first and second microcolumns 112, 212 is to be employedso as to allow pressurizing the upper or first microcolumns 112 from thetop without yet passing the liquid through to the lower or secondmicrocolumns 212, more about which is said below. Notably, theconfiguration of the exit 22 or tip region 33 and/or the reduceddiameter bed region 31 (FIG. 1A) of the first microcolumn 112 is suchthat there is a loose fit or gaps left when nesting in the entrance 20at the full diameter bed region 30 of the second microcolumn 212 so asto allow venting and thus pressurization from above and activation ofthe first microcolumn 112 without necessarily activating, pressurizing,or flowing through the second microcolumn 212.

Referring to FIG. 10B, there is shown an enlarged partialcross-sectional view of an upper or first microcolumn 112 associatedwith the upper or first stage or array 170 of the apparatus 110, astaken along section line 10B-10B of FIG. 10A. Similar to the exemplaryembodiments of FIGS. 8A and 8B, the first microcolumn 112 is shown ashaving a first or upper extraction system or layered constructionlocated in the upper full diameter bed region 130 but no extractionsystem or layers in the lower reduced diameter bed region 131.Specifically, the upper first layered construction is comprised of: (i)upper flow distributor 116 a configured as a screen or mesh, (ii) uppercompression layer 118 a such as a glass fiber filter material, (iii)optional sorbent or extraction layer 119, and (iv) optional frit 117.Again, additional layers may be added or substituted, it being noted,for example, the alternative position of the optional frit 117 at thebottom of the layers rather than at an intermediate position and of anupper sorbent 119 rather than a second flow distributor layer (compareto middle flow distributor 16 b of FIGS. 1B, 3, 7A, 7B, 8A and 8B).

And in FIG. 10C there is shown an enlarged partial cross-sectional viewof a lower or second microcolumn 212 associated with the lower or secondstage or array 270 of the apparatus 110 shown in FIG. 10A, as takenalong section line 10C-10C of FIG. 10A. Similar to the exemplaryembodiments of FIGS. 9A and 9B, the second microcolumn 212 is shown ashaving a second or lower extraction system or layered constructionlocated in the lower reduced diameter bed region 231 but no extractionsystem or layers in the upper full diameter bed region 230.Specifically, the lower second layered construction is comprised of: (i)middle compression layer 218 c, (ii) extraction layer 214 ofmicroparticulate extraction medium, and (iii) lower compression layer218 b. Again, additional layers may be added or substituted, here itbeing noted, for example, that the lower and middle compression layers218 b, 218 c may be frits or any other such materials bounding theextraction layer 214 and that the optional lower flowdistributor/support 16 c (FIGS. 9A and 9B) which may be molded as partof the container is not included in the alternative exemplary lower orsecond microcolumn 212 associated with the second stage or array 270 ofthe apparatus 10. As also shown in FIG. 10C, optionally, microcolumnpassage 223 may be tapered from the upper full diameter bed region 230to the reduced diameter bed region 231, which it will be appreciatedassists in transitioning the sample or elution liquid from one bedregion to the other.

More generally, with continued reference to FIGS. 10A-10C, it will beappreciated that by configuring the apparatus 110 in two stages with twoseparate microcolumns 112, 212 in series, the upper first microcolumn112 being configured with a layered construction in its upper fulldiameter bed region 130 and the lower second microcolumn 212 beingconfigured with a layered construction in its lower reduced diameter bedregion 231, there is achieved a multi-stage extraction system somewhatanalogous to doing so in a single microcolumn 12 having two distinct bedregions and related layered constructions, or effectively two stages,but by doing so in two microcolumns 112, 212 in series more flexibilityin use is beneficially derived. For example, by physically separatingthe upper and lower extraction or filtration systems between the twomicrocolumns 112, 212, larger volumes or spaces therebetween are formed,though with less dead spaces, both to serve as functional reservoirs forsamples or elution liquids and effectively forming air gaps to preventor mitigate against dripping or capillary flow and to allow for thetransfer of the one or more columns between each other or to anappropriate receiving vessel or plate. In that regard, there is thusprovided new functional utility and increased fields of use. In oneembodiment, such a single- or multi-stage microcolumn alone or in anarray functions as a test tube or incubation device, wherein aself-contained system is provided in which aqueous solutions will notreadily leak out based on supported surface tension and other suchfactors yet with the ability to also serve as an effective sold phaseextraction device. Furthermore, in the particular exemplary embodimentwherein the upper or first microcolumn 112 associated with the firststage comprises the optional sorbent or extraction layer 119, it will beappreciated that there is thus provided a true dual- or two-stage solidphase extraction apparatus 110. Those skilled in the art will appreciatethat a variety of other arrangements of the microcolumns 112, 212 andthe layered constructions within each are possible without departingfrom the spirit and scope of the invention. By way of furtherillustration and not limitation, when the first and second microcolumns112, 212 are configured as set forth herein so as to nest in an unsealedor vented fashion, it will be appreciated that when a sample is firstfiltered or extracted in the upper or first stage microcolumns 112pressure is applied thereto from above so as to in this example push thesample through the first or upper extraction system or layeredconstruction located in the upper full diameter bed region 130 of eachand have the sample flow downwardly and collect within the passage 223of the respective lower or second microcolumns 212 above the second orlower extraction system or layered construction located in the lowerreduced diameter bed region 231 of each lower second microcolumn 212without flowing or being forced therethrough, again because the pressureapplied to the upper first microcolumns 112 is vented. Once the sampleis prepared as through filtration or extraction via the firstmicrocolumns 112, the upper or first array 170 of microcolumns 112 maysimply be removed or jettisoned and then further processing conducted onthe lower or second array 270 of microcolumns 212, as by thenpressurizing the second microcolumns 212 and/or employing an elutionsolvent to release the analytes of interest. It will be appreciated thata variety of mechanisms for manually, semi-automatically orautomatically selectively activating or filtering with the upper firstarray 170 of microcolumns 112 and then the lower second array 270 ofmicrocolumns 212, whether now known or later developed, are possibleaccording to aspects of the present invention, which will be furtherappreciated by the further discussion and examples herein. It will alsobe appreciated that while the second stage filtration or extractionactivation may be accomplished using pressure, centrifuge or gravity orother such techniques now known or later developed to draw the samplethrough the second microcolumns 212 may also be employed withoutdeparting from the spirit and scope of the present invention.

Referring next to FIGS. 11A-11C there is shown a still further exemplaryembodiment of an apparatus 110 for extracting an analyte from a liquidsample similar to that of FIGS. 10A-10C with the apparatus 110 againconfigured as a multi-column array or format (aka, an “extractionplate”) of microcolumns 112, 212 in series as by stacking or nesting onearray of microcolumns on or over another. Once more, there are thusformed an upper first array 170 of first microcolumns 112 and a lowersecond array 270 of second microcolumns 212. In the illustratedembodiment, each array 170, 270 comprises a plurality of microcolumns112, 212 maintained in a substantially parallel and offset pattern orarrangement as by a respective top plate 160, 260 and base 162, 262—asagain illustrated, there are ninety-six (96) such microcolumns 112, 212per array 170, 270, though it will be appreciated that such is merelyexemplary. The nesting of respective first and second microcolumns 112,212 may again be achieved employing any appropriate technique now knownor later developed, including sealed (tight) and unsealed (loose) fitarrangements, though there again being advantages in the presentmulti-stage arrangement and micro elution bed configuration to looselynesting the respective microcolumns 112, 212 so as to vent therebetween.

As shown in the enlarged partial cross-sectional view of an upper orfirst microcolumn 112 associated with the upper or first stage or array170 of the apparatus 110 of FIG. 11B, here as taken along section line11B-11B of FIG. 11A, similar to the exemplary embodiments of FIGS. 8Aand 8B and FIG. 10B, the first microcolumn 112 is shown as having afirst or upper extraction system or layered construction located in theupper full diameter bed region 130 comprised of: (i) upper flowdistributor 116 a configured as a screen or mesh, (ii) upper compressionlayer 118 a such as a glass fiber filter material, (iii) optionalsorbent or extraction layer 119, and (iv) optional frit 117.Furthermore, the upper or first microcolumn 112 also includes anadditional extraction system or layered construction in the lowerreduced diameter bed region 131, here shown much like that of theembodiment of FIG. 7B wherein there is provided: (i) middle compressionlayer 118 c, (ii) air gap layer 114 (rather than an extraction layer 14as in FIGS. 1B and 3), and (iii) lower compression layer 118 b. It willbe appreciated that the space between the upper extraction system orlayered construction, here including an optional lowest layer as frit117, and the top of the lower extraction system or layered construction,here being the middle compression layer 118 c, defines yet another airgap layer analogous to the air gap layer 301 of FIG. 7B. Thus, there areeffectively two air gap layers in the upper or first microcolumn 112alone in this alternative exemplary embodiment. Once again, thoseskilled in the art will appreciate that the number, arrangement, andactual or proportional size of any such air gaps are merely illustrativeof aspects of the present invention and non-limiting. It is again notedin connection with FIG. 11B that, though not shown, an optional lowerflow distributor/support analogous to such distributor 16 c shown inFIG. 7B may be molded as part of the container or otherwise provided aspart of the lower reduced diameter bed region 131 of the upper or firstmicrocolumn 112. Again, additional layers may be added or substituted,it being noted, for example, the alternative position of the optionalfrit 117 and the inclusion of an upper sorbent 119 rather than a secondflow distributor layer (compare to middle flow distributor 16 b of FIGS.1B, 3, 7A, 7B, 8A and 8B).

And in FIG. 11C there is again shown an enlarged partial cross-sectionalview of a lower or second microcolumn 212 associated with the lower orsecond stage or array 270 of the apparatus 110 shown in FIG. 11A, astaken along section line 11C-11C of FIG. 11A. For simplicity, the secondmicrocolumn 212 of FIG. 11C is shown as being identical to that of FIG.10C, though it will be appreciated that other such configurations of thelower or second extraction system or layered construction located in thelower reduced diameter bed region 231, with or without an extractionsystem or layers in the upper full diameter bed region 230, may beemployed without departing from the spirit and scope of the invention.In the illustrated embodiment, once again, the lower second layeredconstruction is comprised of: (i) middle compression layer 218 c, (ii)extraction layer 214 of microparticulate extraction medium, and (iii)lower compression layer 218 b, with additional layers being added orsubstituted depending on the context. As also again shown in FIG. 11C,optionally, microcolumn passage 223 may be tapered from the upper fulldiameter bed region 230 to the reduced diameter bed region 231 to assistin transitioning the sample or elution liquid from one bed region to theother.

Regarding the alternative embodiment of the apparatus 110 as shown inFIGS. 11A-11C, it will again be appreciated that there are provided twostages with two separate microcolumns 112, 212 in series, the upperfirst microcolumn 112 being configured with a layered construction atleast in its upper full diameter bed region 130 and the lower secondmicrocolumn 212 being configured with a layered construction in itslower reduced diameter bed region 231. Accordingly, there is once moreachieved a multi-stage extraction system as generally shown anddescribed herein with at least two distinct bed regions and relatedlayered constructions, or effectively two stages, again doing so in twomicrocolumns 112, 212 in series. As noted above in connection with thealternative upper or first microcolumn 112 of the present embodiment, byincluding spaced apart middle and lower compression layers 118 c, 118 bthere are provided two air gaps in the first microcolumn 112 alone, andagain, by physically separating the upper and lower extraction orfiltration systems between the two microcolumns 112, 212, larger volumesor spaces therebetween are formed that further serve as air gaps, allsuch air gaps cooperating to prevent or mitigate against dripping orcapillary flow and to allow for the transfer of the one or more columnsbetween each other or to an appropriate receiving vessel or plate.Moreover, in the exemplary arrangement of the upper or first microcolumn112 as shown in FIG. 11B, a further alternative embodiment might be toinclude an extraction layer or sorbent analogous to extraction layer 14in FIGS. 1B, 3 and 9A rather than the air gap 114 shown in FIG. 11B. Itwill be appreciated that the result in this alternative exemplaryembodiment would be effectively three extraction systems or athree-stage solid-phase extraction system achieved using two of thenovel microcolumns 112, 212 in series according to aspects of thepresent invention, with two such stages occurring in the upper firstmicrocolumn 112 and an effective third stage in the lower secondmicrocolumn 212. It will be further appreciated that a four-stagearrangement could be provided by also including an extraction system inthe upper full diameter bed region 230 of the lower or secondmicrocolumn 212, or there may be only one stage in the upper firstmicrocolumn 112 as shown in FIGS. 10B and 11B and instead two stages inthe lower second microcolumn 212. Again, those skilled in the art willappreciate that any such arrangements or configurations are possiblewithout departing from the spirit and scope of the invention, includinghaving two or more stages and two or more microcolumns in series, withthe stages distributed among the microcolumns in a wide variety ofarrangements. In that regard, there is once more thus provided newfunctional utility and increased fields of use in the extractionapparatus 110 according to aspects of the present invention, wherebyrelatively more effective extraction as measured by absolute recovery orotherwise is possible even using the same or smaller elution volume,resulting in less dead or unused volume and less contaminates in theextracted analytes.

Although the present invention has been described in considerable detailwith reference to certain preferred versions thereof, other versions arepossible. For example, the apparatus 10 is not limited to use withbiological fluids, but can be used, for example, for testing groundwater, drinking water, and other liquids for contaminants.

Aspects of the present specification may also be described as follows:

-   1. An apparatus for extracting an analyte from a liquid sample    comprising: a) a container having an entrance, an exit, and a    passage therebetween for passage of a liquid sample containing an    analyte therethrough, said container having a full diameter bed    region and a reduced diameter bed region; b) within the passage in    the reduced diameter bed region, a thin layer of microparticulate    extraction media, wherein the extraction media layer has a top    surface, a bottom surface, and a peripheral edge, and the extraction    media layer is oriented in the passage so that liquid flows through    the extraction media layer from its top surface to the bottom    surface; c) an upper compression layer in the full diameter bed    region having an effective diameter bed area, located at or spaced    above the top surface of the extraction media layer and a lower    compression layer in the reduced diameter bed region, located at or    spaced below the bottom surface of the extraction media layer, the    two compression layers pressing the extraction media therebetween,    wherein the effective area of the of the extraction media is smaller    than the effective area of the upper compression layer; and d) an    upper mesh flow distributor above the upper compression layer for    distributing flow of the liquid sample uniformly to the extraction    media layer top surface.-   2. The apparatus of embodiment 1, wherein the ratio between the    effective area of the extraction media to the effective area of the    upper compression layer is about 1:10.-   3. The apparatus of embodiment 1 or embodiment 2, wherein the ratio    between the effective area of the extraction media to the effective    area of the upper compression layer is about 1:4.-   4. The apparatus of any one of embodiments 1-3, wherein the    extraction media has a number average particle size of about less    than 20 μm.-   5. The apparatus of any one of embodiments 1-4, wherein the    extraction media has a small particle size having a number average    particle size of less than about 40 microns, less than about 30    microns, less than about 25 microns, less than about 20 microns,    less than about 15 microns, less than about 10 microns, or less than    about 5 microns.-   6. The apparatus of any of embodiments 1-5, wherein the extraction    media comprises particles that are sorbent particles selected from    ion exchange sorbents, reversed phase sorbents, and normal phase    sorbents.-   7. The apparatus of embodiment 6, wherein the sorbent particles are    selected from an inorganic material such as SiO₂ or an organic    polymeric material such as poly(divinylbenzene), silica based    particles (such as silica based carboxylic acids), diatomaceous    earth particles, polymeric based particles, mono-dispersed silica    and polymeric particles, and/or carbon graphite particles.-   8. The apparatus of embodiment 7, wherein the sorbent particles are    treated with an organic functional group such as a C₂-C₂₂,    preferably C₈-C₁₈, functional group.-   9. The apparatus of any of embodiments 1-8, wherein the extraction    bed comprises at least two different extraction media in a single    bed.-   10. The apparatus of any of embodiments 1-9, wherein the apparatus    further comprises one or more additional extraction beds.-   11. The apparatus of any of embodiments 1-10, wherein the extraction    media is loosely packed or is compacted.-   12. The apparatus of any of embodiments 1-11, wherein one or more of    the compression layers is flat, spherical, a truncated cone,    prismatic, or a truncated pyramid.-   13. The apparatus of any of embodiments 1-12, wherein one or more of    the compression layers has a pore size less than the particle size    of the extraction media.-   14. The apparatus of embodiment 13, wherein one or more of the    compression layers has a pore size of less than 5 microns or less    than 3 microns.-   15. The apparatus of any of embodiments 1-14, wherein the    compression layers are composed of a flexible, hydrophobic material.-   16. The apparatus of embodiment 15, wherein the compression layer    comprises a glass microfiber media and/or a polymer.-   17. The apparatus of embodiment 15, wherein the compression layer    comprises polypropylene or polyethylene.-   18. The apparatus of any of embodiments 1-17, wherein the    compression layer has a thickness ranging from about 0.1 mm to about    3.25 mm, about 0.25 mm to about 3.25 mm, about 0.5 mm to about 3.0    mm, about 0.75 mm to about 3 mm, about 0.25 mm to about 2.5 mm,    about 0.25 mm to about 2 mm, about 0.25 mm to about 1.5 mm, about    0.25 mm to about 1.25 mm, about 0.25 mm to about 1.0 mm, about 0.1    mm to about 0.75 mm, or about 0.1 mm to about 0.5 mm.-   19. The apparatus of any of embodiments 1-18, wherein the flow    distributor is a flexible mesh material having a mesh number of 200    or higher.-   20. The apparatus of any of embodiments 1-19, wherein the flow    distributor comprises one or more of Polyphenelyne Sulfide (PPS),    Polytetrafluoroethylene (PTFE), Polyether ether Ketone (PEEK),    Polyoxymethylene (POM), Ethylene Propylene Diene (EPDM), a    Fluorinated elastomer (FKM), a Perfluoro elastomer (FFKM),    polysulfone (PSU), Ethylene Tetrafluoroethylene (ETFE),    Polypropylene (PP), (Poly)Chlorotrifluoroethylene (PCTFE/CTFE),    polystyrene, high density polyethylene, polycarbonate, nylon,    polyethylene terephthalate (PET), silicon, rubber, or polyester.-   21. The apparatus according to any of embodiments 1-20, further    comprising a middle flow distributor below the upper compression    layer and/or a lower flow distributor below the lower compression    layer.-   22. The apparatus according to embodiment 21, wherein the flow    distributors are layered or molded in the housing above and below    the compression layers.-   23. The apparatus according to embodiment 22, wherein the upper flow    distributor is located in the full diameter region and the lower    flow distributor is seated in a lower portion of the apparatus,    having a reduced diameter.-   24. The apparatus according to any of embodiments 1-23, wherein the    apparatus is configured for an elution volume of about 0.025 ml to    about 0.25 ml, about 0.025 ml to about 0.2 ml, about 0.025 ml to    about 0.15 ml, about 0.025 ml to about 0.100 ml.-   25. The apparatus according to any of embodiments 1-24, wherein the    entrance is configured to receive a connective fitting to connect    the apparatus to a fluid input device.-   26. The apparatus according to any of embodiments 1-25, wherein the    fluid input device comprises automated fluid dispensing systems,    automated liquid handling platforms, syringes, and microdispensers.-   27. An extraction plate comprising multiple apparatuses according to    any of embodiments 1-26.-   28. The extraction plate according to embodiment 27, wherein the    apparatuses are arranged in a multicolumn array.-   29. An apparatus for extracting an analyte from a liquid sample    comprising: a) a container having an entrance, an exit, and a    passage therebetween for passage of a liquid sample containing an    analyte therethrough, said container having a full diameter bed    region and a reduced diameter bed region; and b) a layered    construction having a top and a bottom extending across the passage,    comprising from top to bottom: (i) an upper flow distributor/support    layer, (ii) an upper compression layer, (iii) an extraction layer of    microparticulate extraction medium adjacent to the layer (ii),    and (iv) a lower compression layer located adjacent to the    extraction layer (iii), wherein the (i) upper flow distributer    and (ii) upper compression layer are located in the full diameter    region, the (iii) extraction layer and (iv) lower compression layer    are located in the reduced diameter region.-   30. The apparatus of embodiment 29, wherein the full diameter region    has an effective bed area measured by A_(F)=πr_(F) ², where r_(F) is    the radius of an interior surface of the container in the full    diameter region, and A_(r)=πr_(r) ^(r) where r_(r) is the radius of    an interior surface of the container in the reduced diameter region,    and wherein the ratio between the effective bed area of the full    diameter region and the effective area of the reduced diameter    region ranges from about 10:1 to 1.5:1.-   31. The apparatus of embodiment 29, wherein the ratio between the    effective bed area of the full diameter region and the effective    area of the reduced diameter region is about 4:1.-   32. The apparatus of embodiment 29, wherein the layered construction    having a top and a bottom extending across the passage, comprises    from top to bottom: (i) an upper flow distributor, (ii) an upper    compression layer, (i′) a middle flow distributor, (ii′) a middle    compression layer in the reduced diameter region, (iii) an    extraction layer of microparticulate extraction medium adjacent to    the layer (ii′), (iv) a lower compression layer adjacent to layer    (iii), and (v) optionally, a lower flow distributor, wherein layers    (i), (ii), (i′), and (ii′) are located in the full diameter region,    and layers (iii)-(v) may be located in the reduced diameter region.-   33. The apparatus of embodiment 29, wherein the layered construction    having a top and a bottom extending across the passage, comprises    from top to bottom: (i) an upper flow distributor, (ii) an upper    compression layer, (i′) a middle flow distributor, (ii′) a middle    compression layer in the reduced diameter region, (iii) an    extraction layer of microparticulate extraction medium adjacent to    the layer (ii′), (iv) a lower compression layer adjacent to layer    (iii), and (v) optionally, a lower flow distributor, wherein layers    (i), (ii), and (i′), are located in the full diameter region, and    layers (ii′)-(v) are located in the reduced diameter region.-   34. The apparatus of embodiment 29, further comprising one or more    air gap layer(s).-   35. The apparatus of embodiment 34, wherein an air gap layer is    located in the reduced-diameter region.-   36. The apparatus of embodiment 35, wherein the air gap layer has a    height that ranges from ½ the diameter of the reduced diameter    region to 4 times the diameter of the reduced diameter region.-   37. The apparatus of embodiment 33, further comprising an air gap    layer located between layer (i′) and layer (ii′).-   38. The apparatus of embodiment 37, wherein the air gap layer is    located in the reduced diameter region.-   39. The apparatus according to any of embodiments 34-38, wherein the    air gap has a height range from about 0.5 to about 1 times, about    0.5 to about 1.5 times, about 0.5 to about 2 times, about 96 to    about 2.5 times, about 0.5 to about 3 times, about 0.5 to about 3.5    times, about 96 to about 4 times, about 1 to about 1.5 times, about    1 to about 2 times, about 1 to about 2.5 times, about 1 to about 3    times, about 1 to about 3.5 times, about 1 to about 4 times, about    1.5 to about 2 times, about 1.5 to about 2.5 times, about 1.5 to    about 3 times, about 1.5 to about 3.5 times, about 1.5 to about 4    times, about 2 to about 2.5 times, about 2 to about 3 times, about 2    to about 3.5 times, about 2 to about 4 times, about 2.5 to about 3    times, about 2.5 to about 3.5 times, about 2.5 to about 4 times,    about 3 to about 3.5 times, about 3 to about 4 times, or about 3.5    to about 4 times, the diameter of the reduced diameter region.-   40. The apparatus of any of embodiments 1-26 or embodiments 29-39,    further comprising a tip region.-   41. The apparatus according to embodiment 40, wherein the tip region    is cylindrical or conical.-   42. The apparatus according to embodiment 41, where in the tip    region has the same diameter of the reduced diameter bed region or a    smaller diameter bed than the reduced diameter bed region.-   43. The apparatus according to any of embodiments 40-42, wherein the    tip region is in the form of a luer tip.-   44. An apparatus for extracting an analyte from a liquid sample    comprising: a) a microcolumn having an entrance, an exit, and a    passage therebetween for passage of a liquid sample containing an    analyte therethrough, the microcolumn having a full diameter bed    region and a reduced diameter bed region; and b) an extraction    system having a top and a bottom extending across the passage, the    extraction system comprising at least one of: a first layered    construction comprising from top to bottom: (i) an upper flow    distributor layer and (ii) an upper compression layer, the first    layered construction located in the full diameter bed region; and a    second layered construction comprising from top to bottom: (iii) a    middle compression layer and (iv) a lower compression layer, the    second layered construction located in the reduced diameter bed    region.-   45. The apparatus of embodiment 44 wherein the extraction system    comprises only the first layered construction.-   46. The apparatus of embodiment 44 or embodiment 45 wherein the    first layered construction further comprises a middle flow    distributor layer positioned beneath the upper compression layer.-   47. The apparatus of embodiment 46 wherein the first layered    construction further comprises a frit positioned between the middle    flow distributor layer and the upper compression layer.-   48. The apparatus of any of embodiments 44-47 wherein the first    layered construction further comprises a frit positioned beneath the    upper compression layer.-   49. The apparatus of any of embodiments 44-48 wherein the first    layered construction further comprises an extraction layer beneath    the upper compression layer.-   50. The apparatus of embodiment 44 wherein the extraction system    comprises only the second layered construction.-   51. The apparatus of any of embodiments 44-50 wherein the second    layered construction further comprises an extraction layer of    microparticulate extraction medium between the middle compression    layer and the lower compression layer.-   52. The apparatus of any of embodiments 44-51 wherein the second    layered construction further comprises an air gap between the middle    compression layer and the lower compression layer.-   53. The apparatus of any of embodiments 44-52 wherein the second    layered construction further comprises a lower flow distributor    beneath the lower compression layer.-   54. The apparatus of any of embodiments 44-53 wherein an air gap is    formed between the upper compression layer of the first layered    construction and the middle compression layer of the second layered    construction.-   55. The apparatus of embodiment 44 wherein the extraction system    comprises at least one first layered construction and at least one    second layered construction.-   56. The apparatus of any of embodiments 44-55 further comprising    multiple microcolumns in an array.-   57. The apparatus of any of embodiments 44-56 further comprising    multiple microcolumns in series so as to define at least a first    microcolumn associated with a first stage and a second microcolumn    associated with a second stage.-   58. The apparatus of embodiment 57 wherein the first microcolumn    engages the second microcolumn with unsealed nesting.-   59. An apparatus for extracting an analyte from a liquid sample    comprising: a) an upper first microcolumn and a lower second    microcolumn selectively in series, each microcolumn having an    entrance, an exit, and a passage therebetween for passage of a    liquid sample containing an analyte therethrough, whereby the first    microcolumn defines a first passage and the second microcolumn    defines a second passage, each microcolumn further having a full    diameter bed region and a reduced diameter bed region; b) a first    layered construction having a top and a bottom extending across the    first passage of the first microcolumn, comprising from top to    bottom: (i) an upper flow distributor/support layer and (ii) an    upper compression layer, the first layered construction located in    the full diameter bed region of the first microcolumn; and c) a    second layered construction having a top and a bottom extending    across the second passage of the second microcolumn, comprising from    top to bottom: (iii) an extraction layer of microparticulate    extraction medium and (iv) a lower compression layer, the second    layered construction located in the reduced diameter bed region of    the second microcolumn.-   60. The apparatus of embodiment 59 wherein the first layered    construction further comprises an extraction layer beneath the upper    compression layer.-   61. The apparatus of embodiment 59 or embodiment 60 comprising a    third layered construction having a top and a bottom extending    across the first passage of the first microcolumn, comprising from    top to bottom: (i) a middle compression layer and (ii) a lower    compression layer, the third layered construction located in the    reduced diameter bed region of the first microcolumn.-   62. The apparatus of embodiment 61 wherein the third layered    construction further comprises an air gap between the middle    compression layer and the lower compression layer.-   63. The apparatus of embodiment 61 or embodiment 62 wherein the    third layered construction further comprises an extraction layer    between the middle compression layer and the lower compression    layer.-   64. A method for the extraction of an analyte from a sample    comprising: a) contacting an apparatus or extraction plate of any of    embodiments 1-63 with a sample in a liquid buffer, and b) eluting    the sample from the apparatus with an elution buffer.-   65. The method according to embodiment 64, further comprising one or    more conditioning or washing steps before, during, or after    contacting the apparatus with the sample.-   66. The method of embodiment 64 or embodiment 65 wherein the step of    contacting the apparatus comprises filtering the sample through the    first microcolumn under pressure and flowing the sample into the    second microcolumn under reduced pressure as by unsealingly nesting    the first microcolumn within the second microcolumn so as to provide    ventilation.-   67. The method of any of embodiments 64-66 wherein the step of    contacting the apparatus further comprises removing the first    microcolumn and filtering the sample through the second microcolumn    and the step of eluting the sample from the apparatus comprises    flowing the elution buffer through the second microcolumn.-   68. A kit comprising an apparatus or extraction plate according to    any of embodiments 1-63.-   69. The kit according to embodiment 68, further comprising one or    more elution buffers or wash buffers.

EXAMPLES

The following non-limiting examples are provided for illustrativepurposes only in order to facilitate a more complete understanding ofthe disclosed subject matter. These examples should not be construed tolimit any of the embodiments described in the present specification,including those pertaining to the apparatus used therein and the methodsof using the present apparatus.

Example 1 Extraction of the Catecholamines from Plasma

Control Single diameter column: CEREX® WCX 1 cc 10 mg columns.

Present Apparatus “narrow bore column”: The narrow bore column was a 1cc column having an effective area ratio of 4:1 (full diameter toreduced diameter). The layers of the column were: (i) an upper flowdistributor screen, (ii) a cylindrical fabric compression layer, (iii) alower flow distributor screen, (iv) a spherical frit as a compressionlayer, (v) the microparticulate extraction medium, and (vi) a sphericalfrit as a lower compression layer. Layers (i)-(iii) were located in thefull diameter region, where layers (iv)-(vi) were located in the reduceddiameter region. The extraction medium was the same as the extractionmedium used in the CEREX® WCX 1 cc 10 mg columns.

Normal human serum samples were obtained from Bioreclamation Corp. andspiked with catecholamines prior to solid phase extraction. The blanksand plasma samples were also spiked with internal standards (e.g.,dopamine-D4, epinephrine-D6, and norepinephrine-D6).

CEREX® WCX (1 cc/10 mg) (control and narrow bore) columns wereconditioned with 0.5 ml of methanol, followed by 0.5 ml of 10 mMPhosphate Buffer pH 6.8.

0.5 mL 10 mM Phosphate buffer was mixed with 100 μL of the Sample. TheSample/buffer mix at a pH of 6.8 was loaded onto the column at apressure of 2-3 psi. The column was washed with 1 mL deionized water at6 psi and subsequently washed with 1 ml Acetonitrile at 6 psi.

Two sets of columns were loaded for each type of column. One half of thecolumns (control and narrow bore) were eluted with 0.5 mL of ElutionBuffer—25:75 100 mM Potassium Carbonate:Acetonitrile. The other half ofthe columns were eluted with 0.1 mL of 25:75 100 mM PotassiumCarbonate:Acetonitrile.

25 μL of the eluent was used or loaded for the LC-MS/MS reaction.

Example 2 LC-MS/MS Analysis of the Catecholamines

25 μL of the solution obtained from the extraction was automaticallyinjected into a TARGA® C18 3 μm particle size 50×2.1 mm analyticalcolumn. A binary HPLC gradient was applied to the analytical column toseparate the epinephrine, norepinephrine and dopamine from otheranalytes contained in the sample. Mobile phase A was 5.0 mM ammoniumformate with 0.1% formic acid pH 3.0 and mobile phase B was Acetonitrilewith 0.1% formic acid. The HPLC gradient proceeded at a temperature of35° C. with a flow rate of 500 μL/min over five minutes as follows:

Gradient: Time (min) B (%) 0.01 50 3.00 100 4.00 100 4.50 50 5.00 50

MS/MS was performed using an APPLIED BIOSYSTEMS MDS SCIEX 500®, althoughother suitable MS/MS apparatuses are known. The following softwareprograms were used in the present examples: ANALYST 1.5.20, althoughother suitable software systems are known. Liquid solvent/analyteexiting the analytical column flowed to the heated nebulizer interfaceof the MS/MS analyzer. The solvent/analyte mixture was converted tovapor in the heated tubing of the interface. Analytes in the nebulizedsolvent were ionized by heated ESI.

Ions passed to the first quadrupole (Q1), which selected ions with amass to charge ratio of parent ions generated from one of the analytes.Ions entering quadrupole 2 (Q2) collided with argon gas to generate ionfragments, which were passed to quadrupole 3 (Q3) for further selection.After measurement of ions indicative of one of the analytes, Q1 wasadjusted so that ions with a mass to charge ratio of parent ion from asecond analyte were selected. These ions were collided with nitrogen gasin Q2, and the ion fragments passed to Q3 for further selection. Aftermeasurement of these ions, Q1 was adjusted so that ions with a mass tocharge ratio of parent ion from a third analyte were selected. Theseions were collided with argon gas in Q2, and the ion fragments passed toQ3 for further selection. Simultaneously, the same process using isotopedilution mass spectrometry was carried out with internal standards,dopamine-D4 and/or epinephrine-D6, and/or norepinephrine-D6. Thefollowing mass transitions were used for detection and quantitation ofepinephrine, norepinephrine and dopamine (and their correspondinginternal standards) during validation on positive polarity from the samesample injection.

Table 1 shows the data representing the percent recovery ofcatecholamines from a plasma sample spiked with 1 ng/ml catecholamine.Notably, the narrow bore columns performed significantly better for allcatecholamines at all elution volumes. In particular, the small volumeelution (i.e., the 0.1 ml elution) performed at least approximatelythree times as well as the small volume elution using the conventionalcolumn.

TABLE 1 Absolute Recovery Comparison at 1 ng/ml Plasma Cerex 10 mg 1 ccCerex 10 mg 1 cc Cerex 10 mg 1 cc Cerex 10 mg 1 cc WCX column WCX columnWCX Narrow Bore WCX Narrow Bore Compound 0.5 ml elution 0.1 ml elution0.5 ml elution 0.1 ml elution Dopamine 98.3% cv = 8.3%  27.6% cv = 11.5%99.5% cv = 4.6% 99.6% cv = 4.4% Dopamine-D4 96.8% cv = 9.8%  32.4% cv =10.4% 99.5% cv = 4.2% 98.6% cv = 4.7% Epinephrine 92.3% cv = 12.6% 35.6%cv = 11.5% 97.5% cv = 3.8% 97.2% cv = 5.0% Epinephrine-D6 91.4% cv =12.8% 38.5% cv = 13.2% 96.7% cv = 4.1% 98.1% cv = 4.5% Norepinephrine87.3% cv = 14.3% 15.6% cv = 22.5% 93.0% cv = 5.8% 92.1% cv = 6.2%Norepinehrine-D6 85.6% cv = 15.2% 13.9% cv = 23.0% 91.5% cv = 5.4% 90.9%cv = 5.7%

As shown in the chromatograms of FIG. 4A-F and FIG. 5A-F, extracting anddetecting catecholamine in plasma from healthy donors by using thepresent apparatus (i.e., the narrow bore column) is plotted over time.

The chromatography demonstrates the sensitivity of the assay. Theabsolute recovery chart demonstrates the improved recovery and improvedreproducibility using the narrow bore columns vs conventional columns,even at smaller elution volumes.

Another benefit of the present apparatus is that due to the smallerelution volumes from the reduced effective bed diameter the presentapparatus can collect into smaller vessels and eliminate or reducetransfers.

Example 3 Extraction of Buprenorphine and Norbuprenorphine from Urine

Narrow Bore Extraction columns feature high-capacity, high-efficiency,low bed mass sorbents that permit the use of low elution volumes (50-100μL). These elution volumes lend themselves to evaporation within thepositive pressure SPE processor unit such as an ALDIII™ or an IP8™(SPEware Corp., Baldwin Park, Calif.), eliminating the need for aseparate solvent evaporator. This allows the use of selective elutionsolvents, resulting in cleaner extracts than would be possible usinghigh solvent strength (low specificity) elution solvents.

Buprenorphine and norbuprenorphine were chosen as model compounds forseveral reasons. They are frequently monitored as part of “pain panels”in compliance testing laboratories. Their relevant concentrations arerelatively low and thus require low LLODs. Additionally, they areexcreted in urine primarily as glucuronide conjugates, affording theopportunity to evaluate both solid phase extraction efficiency withnarrow bore SPE columns.

Experimental Reagents

a) water, b) negative control urine (diluent for the standard curve),and c) a “master mix” containing 100 mM sodium acetate buffer pH 4.8,the internal standard solution (B-d4 and N-d3), and β-glucuronidasesolution (2500 units/sample, catalog #BG100, red abalone, Kura Biotec,Inglewood, Calif.).

Process

A calibration curve was prepared from a single high calibrator viaserial dilution. “Master mix” was placed in all wells of a 96-wellincubation plate on the plate heater (preheated to 68° C.). Calibratorsand controls (100 μL specimen volume) were then transferred to theincubator plate. After 15 minutes, the contents of the incubation platewere transferred to the 96-well SPE plate for extraction.

Solid phase extraction:

Apply samples to narrow bore extraction columns with PSCX sorbent (2.5mg)

Wash w/ 250 μL deionized water

Wash w/ 150 μL 100 mM acetic acid

Wash w/ 300 μL methanol

Dry sorbent for 1 minute

Transfer the SPE plate to collection

Elute w/ 50 μL elution solvent (ethyl acetate: methanol:conc.NH4OH=93:5:2)

Dry the solvent

Dissolve residues in the reconstitution solvent (100 mL, aqueous formicacid 0.1%:methanol=80:20)

Analytical Conditions

LC Conditions:

Column: Haisil C18 HL, 50∴2.1 mm, 5 μM (Higgins Analytical, Inc.,Mountain View, Calif.)

Flow: 400 μL/min

Injection volume: 10 μL

A=0.1% Aqueous formic acid; B=Methanol;

Gradient: 20-40% B in 0.5 min., 40-60% B in 2 min.

MS Conditions: Sciex 5000, Source=ESI; Positive ion MRM

Buprenorphine 468.25→55.10 (quant), 83.2 (qual)

Buprenorphine-d4 472.42→59.0 (quant), 83.0 (qual)

Norbuprenorphine 414.200→55.10 (quant), 83.0 (qual)

Norbuprenorphine-d3 417.01→54.8 (quant), 83.1 (qual)

Results and Conclusions

Calibration curves for B and N are shown in FIG. 6A-B. Regression isquadratic, 1/x weighted. Based on s/n ratios of the quantitation ionsand assessment of curve accuracy (requiring ±20% from nominal value atthe low calibrator), the LLOQs were determined to be 0.313 ng/mL and0.625 for B and N, respectively.

Results from the analysis of non-validated control samples (n=10 each)are as follows:

TABLE 2 Burprenorphine Buprenorphine Hydrolysis Hydrolysis Low Control#1 Low Control #2 Control Low Control #1 Low Control #2 Control 0.75ng/mL 1.2 ng/mL 5.0 ng/mL 100 ng/mL 150 ng/mL 100 ng/mL Average 0.91 1.45.3 107 167 110 Bias  22%  16%   6%   7%  11%  10% CV (RSD) 3.1% 3.1%3.6% 3.2% 4.0% 3.6%

TABLE 3 Norbuprenorphine Norbuprenorphine Hydrolysis Hydrolysis LowControl #1 Low Control #2 Control Low Control #1 Low Control #2 Control0.75 ng/mL 1.2 ng/mL 5.0 ng/mL 100 ng/mL 150 ng/mL 100 ng/mL Average0.97 1.4 4.7 106 165 103 Bias  29%  16%   −6%   6%  10%   3% CV (RSD)3.5% 4.0%   4.8% 2.7% 4.0% 5.6%

RSDs were less than 5%. Absolute recoveries from urine using the NarrowBore Extraction column with a 2.5 mg bed mass were 91% and 97% for B andN, respectively. The experiment was repeated with a urine samplesupplemented with morphine at 20,000 ng per mL. No difference inabsolute recovery was noted.

Example 4 Extraction of Benzoylecgonine, Buprenorphine, EDDP, andMeprobamate from Urine

The following experiment compares extraction recovery of four (4) commonanalytes in urine with regular CEREX HPSCX NBE columns and a set of dualstage SPE columns with filtration only on the first stage and HPSCXsorbent in the narrow bore second stage without top filtration layers.

A blank urine sample was spiked with 100 ng of benzoylecgonine,buprenorphine, EDDP, and meprobamate with their deuterated internalstandards.

Sample Preparation

Regular NBE columns:

Urine sample (50 uL) was mixed with 100 mM sodium acetate (aq., pH 4.8,250 uL) and loaded into the NBE SPE columns. Washing the columns with100 mM HCI (300 uL), then DI water (500 uL), dry the columns with astream of nitrogen for 10 minutes. Elute the sample with the elutionbuffer (DCM:IPA:NH4OH=80:18:2, 50 uL). The eluted samples were driedover nitrogen. The residue was reconstituted with 200 μL of a mixture of0.1% aqueous formic acid:methanol=95:5 and spiked with IS. The sampleswere analyzed via LCMS.

Solid Phase Extraction with Dual Stage NBE Columns:

Urine samples (50 uL) was mixed with 100 mM sodium acetate (aq., pH 4.8,250 uL) and loaded into the dual stage NBE columns. The samples wereapplied to the second stage columns through the first columns. The firststage columns were removed. The samples were loaded onto the sorbent inthe second stage columns. The columns with sorbent were washed with 100mM HCl (300 uL), then DI water (500 uL). Columns were dried through astream of nitrogen for 10 minutes. The residue was reconstituted with100 μL of a mixture of 0.1% aqueous formic acid:methanol=90:10 andskiped with IS. The samples were analyzed via LCMS.

Process

LCMS Method:

Analysis of the extracts was performed on a SCIEX 5500 mass spectrometerinterfaced to a Shimadzu Nexera XR UHPLC. The acquisition program wasbuilt using scheduled MRM (two product ions for each analyte, on production for each internal standard).

TABLE 4 MRM Table: Q1 Q3 DT Transition ID DP EP CP CXP 293.1 171.1 50 ISBenzoylecgonine-D3 200 10 50 12 472.266 400.3 50 IS Buprenorphine-D4 3610 53 18 282.03 235.12 50 IS EDDP-D3 250 10 60 10 226.012 165.2 50 ISMeprobamate-D7 56 10 11 8 290.105 105.1 50 Benzoylecgonine 1 250 10 6010 290.105 119 50 Benzoylecgonine 2 200 10 60 8 468.234 396.1 50Buprenorphine 1 36 10 51 16 468.234 414.2 50 Buprenorphine 2 36 10 45 18279.122 220.1 50 EDDP 1 250 10 75 14 279.122 187 50 EDDP 2 200 10 65 8219.116 158 50 Meprobamate 1 170 10 11 16 219.116 55.2 50 Meprobamate 2140 10 33 14

Analytical Conditions

MS Conditions:

SCIEX QTRAP® 5500, Source=ESI

Positive ion, scheduled MRM

Source temperature 600° C.

Desolvation GS1, GS2: 50

HPLC gradient profile:

Time % B 0.00 5% 4.00 80%  4.20 100%  4.70 100%  4.71 5% 5.71 5%

Mobile Phase A 0.1% Formic acid (aqueous) Mobile Phase B MeOH + 0.1%Formic Acid Flow Rate: 0.7 mL/min Injection Volume 5 ul

LC Conditions:

Shimadzu Nexera XR UHPLC

Column: Raptor™ Biphenyl, 50 mm×2.1 mm, 2.7 um, with Raptor Biphenyl 5mm guard column, Restek Corp., Bellefonte, Pa.

Flow: 700 μL/min

Column temperature: 40° C. (SPEware column oven)

Injection volume: 5 μL

A=0.1% aqueous formic acid; B=0.1% formic acid in Methanol

Results and Conclusions

The following results showed that except for buprenorphine, SPE with thedual stage NBE device provided significantly better recovery than theregular NBE columns with 50 uL of elution solvent.

TABLE 5 Experimental results for four analytes IS % Re- Sample NameComponent Name Area Area covery Std R1 Benzoylecgonine 1 786115 552071100 50 ul elution Benzoylecgonine 1 616656 538764 80 Dual stage HPSCX 50ul elution Benzoylecgonine 1 599230 571348 74 NBE HPSCX Std R1Benzoylecgonine 2 201261 552071 100 50 ul elution Benzoylecgonine 2165762 538764 84 Dual stage HPSCX 50 ul elution Benzoylecgonine 2 163685571348 79 NBE HPSCX Std R1 Buprenorphine 1 351802 304632 100 50 ulelution Buprenorphine 1 302683 234453 112 Dual stage HPSCX 50 ul elutionBuprenorphine 1 343104 254600 117 NBE HPSCX Std R1 Buprenorphine 2320771 304632 100 50 ul elution Buprenorphine 2 288572 234453 117 Dualstage HPSCX 50 ul elution Buprenorphine 2 332709 254600 124 NBE HPSCXStd R1 EDDP 1 1196197 319077 100 50 ul elution EDDP 1 937635 206197 121Dual stage HPSCX 50 ul elution EDDP 1 612008 236699 69 NBE HPSCX Std R1EDDP 2 922362 319077 100 50 ul elution EDDP 2 742065 206197 125 Dualstage HPSCX 50 ul elution EDDP 2 497855 236699 73 NBE HPSCX Std R1Meprobamate 1 609769 477638 100 50 ul elution Meprobamate 1 428435445650 75 Dual stage HPSCX 50 ul elution Meprobamate 1 395076 510502 61NBE HPSCX Std R1 Meprobamate 2 1354992 477638 100 50 ul elutionMeprobamate 2 1046582 445650 83 Dual stage HPSCX 50 ul elutionMeprobamate 2 1008428 510502 70 NBE HPSCX

In closing, it is to be understood that although aspects of the presentspecification are highlighted by referring to specific embodiments, oneskilled in the art will readily appreciate that these disclosedembodiments are only illustrative of the principles of the subjectmatter disclosed herein. Therefore, it should be understood that thedisclosed subject matter is in no way limited to a particularmethodology, protocol, and/or reagent, etc., described herein. As such,various modifications or changes to or alternative configurations of thedisclosed subject matter can be made in accordance with the teachingsherein without departing from the spirit of the present specification.Lastly, the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to limit the scope ofthe present invention, which is defined solely by the claims.Accordingly, the present invention is not limited to that precisely asshown and described.

Certain embodiments of the present invention are described herein,including the best mode known to the inventors for carrying out theinvention. Of course, variations on these described embodiments willbecome apparent to those of ordinary skill in the art upon reading theforegoing description. The inventor expects skilled artisans to employsuch variations as appropriate, and the inventors intend for the presentinvention to be practiced otherwise than specifically described herein.Accordingly, this invention includes all modifications and equivalentsof the subject matter recited in the claims appended hereto as permittedby applicable law. Moreover, any combination of the above-describedembodiments in all possible variations thereof is encompassed by theinvention unless otherwise indicated herein or otherwise clearlycontradicted by context.

Groupings of alternative embodiments, elements, or steps of the presentinvention are not to be construed as limitations. Each group member maybe referred to and claimed individually or in any combination with othergroup members disclosed herein. It is anticipated that one or moremembers of a group may be included in, or deleted from, a group forreasons of convenience and/or patentability. When any such inclusion ordeletion occurs, the specification is deemed to contain the group asmodified thus fulfilling the written description of all Markush groupsused in the appended claims.

Unless otherwise indicated, all numbers expressing a characteristic,item, quantity, parameter, property, term, and so forth used in thepresent specification and claims are to be understood as being modifiedin all instances by the term “about.” As used herein, the term “about”means that the characteristic, item, quantity, parameter, property, orterm so qualified encompasses a range of plus or minus ten percent aboveand below the value of the stated characteristic, item, quantity,parameter, property, or term. Accordingly, unless indicated to thecontrary, the numerical parameters set forth in the specification andattached claims are approximations that may vary. For instance, as massspectrometry instruments can vary slightly in determining the mass of agiven analyte, the term “about” in the context of the mass of an ion orthe mass/charge ratio of an ion refers to +/−0.50 atomic mass unit.

At the very least, and not as an attempt to limit the application of thedoctrine of equivalents to the scope of the claims, each numericalindication should at least be construed in light of the number ofreported significant digits and by applying ordinary roundingtechniques.

Use of the terms “may” or “can” in reference to an embodiment or aspectof an embodiment also carries with it the alternative meaning of “maynot” or “cannot.” As such, if the present specification discloses thatan embodiment or an aspect of an embodiment may be or can be included aspart of the inventive subject matter, then the negative limitation orexclusionary proviso is also explicitly meant, meaning that anembodiment or an aspect of an embodiment may not be or cannot beincluded as part of the inventive subject matter. In a similar manner,use of the term “optionally” in reference to an embodiment or aspect ofan embodiment means that such embodiment or aspect of the embodiment maybe included as part of the inventive subject matter or may not beincluded as part of the inventive subject matter. Whether such anegative limitation or exclusionary proviso applies will be based onwhether the negative limitation or exclusionary proviso is recited inthe claimed subject matter.

Notwithstanding that the numerical ranges and values setting forth thebroad scope of the invention are approximations, the numerical rangesand values set forth in the specific examples are reported as preciselyas possible. Any numerical range or value, however, inherently containscertain errors necessarily resulting from the standard deviation foundin their respective testing measurements. Recitation of numerical rangesof values herein is merely intended to serve as a shorthand method ofreferring individually to each separate numerical value falling withinthe range. Unless otherwise indicated herein, each individual value of anumerical range is incorporated into the present specification as if itwere individually recited herein.

The terms “a,” “an,” “the” and similar referents used in the context ofdescribing the present invention (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. All methods described herein can be performed in any suitableorder unless otherwise indicated herein or otherwise clearlycontradicted by context. The use of any and all examples, or exemplarylanguage (e.g., “such as”) provided herein is intended merely to betterilluminate the present invention and does not pose a limitation on thescope of the invention otherwise claimed. No language in the presentspecification should be construed as indicating any non-claimed elementessential to the practice of the invention.

Specific embodiments disclosed herein may be further limited in theclaims using consisting of or consisting essentially of language. Whenused in the claims, whether as filed or added per amendment, thetransition term “consisting of” excludes any element, step, oringredient not specified in the claims. The transition term “consistingessentially of” limits the scope of a claim to the specified materialsor steps and those that do not materially affect the basic and novelcharacteristic(s). Embodiments of the present invention so claimed areinherently or expressly described and enabled herein.

All patents, patent publications, and other publications referenced andidentified in the present specification are individually and expresslyincorporated herein by reference in their entirety for the purpose ofdescribing and disclosing, for example, the compositions andmethodologies described in such publications that might be used inconnection with the present invention. These publications are providedsolely for their disclosure prior to the filing date of the presentapplication. Nothing in this regard should be construed as an admissionthat the inventors are not entitled to antedate such disclosure byvirtue of prior invention or for any other reason. All statements as tothe date or representation as to the contents of these documents isbased on the information available to the applicants and does notconstitute any admission as to the correctness of the dates or contentsof these documents.

The invention claimed is:
 1. An apparatus for extracting an analyte froma liquid sample comprising: a) an upper first microcolumn and a lowersecond microcolumn selectively in series, each microcolumn having anentrance, an exit, and a passage therebetween for passage of a liquidsample containing an analyte therethrough, whereby the first microcolumndefines a first passage and the second microcolumn defines a secondpassage, each microcolumn further having a full diameter bed region anda reduced diameter bed region; b) a first layered construction having atop and a bottom extending across the first passage of the firstmicrocolumn, comprising from top to bottom: (i) an upper flowdistributor/support layer and (ii) an upper compression layer, the firstlayered construction located in the full diameter bed region of thefirst microcolumn; and c) a second layered construction having a top anda bottom extending across the second passage of the second microcolumn,comprising from top to bottom: (iii) an extraction layer ofmicroparticulate extraction medium and (iv) a lower compression layer,the second layered construction located in the reduced diameter bedregion of the second microcolumn.
 2. The apparatus of claim 1 whereinthe first layered construction further comprises an extraction layerbeneath the upper compression layer.
 3. The apparatus of claim 1comprising a third layered construction having a top and a bottomextending across the first passage of the first microcolumn, comprisingfrom top to bottom: (i) a middle compression layer and (ii) a lowercompression layer, the third layered construction located in the reduceddiameter bed region of the first microcolumn.
 4. The apparatus of claim3 wherein the third layered construction further comprises an air gapbetween the middle compression layer and the lower compression layer. 5.The apparatus of claim 3 wherein the third layered construction furthercomprises an extraction layer between the middle compression layer andthe lower compression layer.
 6. A method for the extraction of ananalyte from a sample comprising: a) contacting an apparatus of claim 1with a sample in a liquid buffer; and b) eluting the sample from theapparatus with an elution buffer.
 7. The method of claim 6 wherein thestep of contacting the apparatus comprises filtering the sample throughthe first microcolumn under pressure and flowing the sample into thesecond microcolumn under reduced pressure as by unsealingly nesting thefirst microcolumn within the second microcolumn so as to provideventilation.
 8. The method of claim 7 wherein the step of contacting theapparatus further comprises removing the first microcolumn and filteringthe sample through the second microcolumn and the step of eluting thesample from the apparatus comprises flowing the elution buffer throughthe second microcolumn.
 9. An apparatus for extracting an analyte from aliquid sample comprising: a first microcolumn comprising a firstpassage, a first full diameter bed region, and a first reduced diameterbed region, a second microcolumn comprising a second passage, a secondfull diameter bed region, and a second reduced diameter bed region, thefirst microcolumn positioned above and selectively in series with thesecond microcolumn with the first passage in fluid communication withthe second passage; a first layered construction extending across thefirst passage and comprising a flow distributor layer and an uppercompression layer, the first layered construction located in the firstfull diameter bed region of the first microcolumn; and a second layeredconstruction extending across the second passage and comprising anextraction layer of microparticulate extraction medium and a lowercompression layer, the second layered construction located in the secondreduced diameter bed region of the second microcolumn.
 10. The apparatusof claim 9 wherein the first passage comprises a first entrance oppositea first exit with the first passage communicating therebetween, thesecond passage comprises a second entrance opposite a second exit withthe second passage communicating therebetween, the first exit in fluidcommunication with the second entrance.
 11. The apparatus of claim 10wherein the first layered construction comprises the flow distributorlayer positioned closer to the first entrance than the upper compressionlayer, the second layered construction comprises the extraction layerpositioned closer to the first entrance than the lower compressionlayer.
 12. The apparatus of claim 9 wherein the first layeredconstruction further comprises a first extraction layer beneath theupper compression layer.
 13. The apparatus of claim 9 further comprisinga third layered construction extending across the first passage andcomprising a middle compression layer and a lower compression layer, thethird layered construction located in the first reduced diameter bedregion of the first microcolumn.
 14. The apparatus of claim 13 whereinthe third layered construction further comprises an air gap between themiddle compression layer and the lower compression layer.